Production of a-Galactosylceramide by a Prominent
Member of the Human Gut Microbiota
Laura C. Wieland Brown1,2., Cristina Penaranda3., Purna C. Kashyap4, Brianna B. Williams1, Jon Clardy2,
Mitchell Kronenberg5, Justin L. Sonnenburg4, Laurie E. Comstock6, Jeffrey A. Bluestone3*,
Michael A. Fischbach1*
1Department of Bioengineering and Therapeutic Sciences and the California Institute for Quantitative Biosciences, University of California, San Francisco, California,
United States of America, 2Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts, United States of America,
3Diabetes Center and the Department of Medicine, University of California, San Francisco, California, United States of America, 4Department of Microbiology and
Immunology, Stanford University School of Medicine, Stanford, California, United States of America, 5La Jolla Institute for Allergy and Immunology, La Jolla, California,
United States of America, 6Division of Infectious Diseases, Department of Medicine, Brigham and Women’s Hospital, Harvard Medical School, Boston, Massachusetts,
United States of America
While the human gut microbiota are suspected to produce diffusible small molecules that modulate host signaling
pathways, few of these molecules have been identified. Species of Bacteroides and their relatives, which often comprise
.50% of the gut community, are unusual among bacteria in that their membrane is rich in sphingolipids, a class of
signaling molecules that play a key role in inducing apoptosis and modulating the host immune response. Although known
for more than three decades, the full repertoire of Bacteroides sphingolipids has not been defined. Here, we use a
combination of genetics and chemistry to identify the sphingolipids produced by Bacteroides fragilis NCTC 9343. We
constructed a deletion mutant of BF2461, a putative serine palmitoyltransferase whose yeast homolog catalyzes the
committed step in sphingolipid biosynthesis. We show that the D2461 mutant is sphingolipid deficient, enabling us to
purify and solve the structures of three alkaline-stable lipids present in the wild-type strain but absent from the mutant. The
first compound was the known sphingolipid ceramide phosphorylethanolamine, and the second was its corresponding
dihydroceramide base. Unexpectedly, the third compound was the glycosphingolipid a-galactosylceramide (a-GalCerBf),
which is structurally related to a sponge-derived sphingolipid (a-GalCer, KRN7000) that is the prototypical agonist of CD1d-
restricted natural killer T (iNKT) cells. We demonstrate that a-GalCerBfhas similar immunological properties to KRN7000: it
binds to CD1d and activates both mouse and human iNKT cells both in vitro and in vivo. Thus, our study reveals BF2461 as
the first known member of the Bacteroides sphingolipid pathway, and it indicates that the committed steps of the
Bacteroides and eukaryotic sphingolipid pathways are identical. Moreover, our data suggest that some Bacteroides
sphingolipids might influence host immune homeostasis.
Citation: Wieland Brown LC, Penaranda C, Kashyap PC, Williams BB, Clardy J, et al. (2013) Production of a-Galactosylceramide by a Prominent Member of the
Human Gut Microbiota. PLoS Biol 11(7): e1001610. doi:10.1371/journal.pbio.1001610
Academic Editor: Philippa Marrack, National Jewish Medical and Research Center/Howard Hughes Medical Institute, United States of America
Received June 11, 2012; Accepted June 6, 2013; Published July 16, 2013
Copyright: ? 2013 Wieland Brown et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This research was supported by NIH grants DP2 OD007290 (to M.A.F.), R37 AI46643 (to J.A.B.), R01 AI044193 (to L.E.C.), R01 AI45053 (to M.K.), R01
GM086258 (to J.C.), F32 (to L.C.W.B.), and P30 DK63720 (for core facilities). The funders had no role in study design, data collection and analysis, decision to
publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
Abbreviations: a-GalCer, a-galactosylceramide; APC, antigen presenting cell; CPE, ceramide phosphorylethanolamine; ELSD, evaporative light scattering
detector; FBS, fetal bovine serum; GF, germ-free; HPLC, high-performance liquid chromatography; HRMS, high-resolution mass spectrometry; iNKT, cell invariant
natural killer T cell; LCB, long-chain base; PBMC, peripheral blood mononuclear cell; PBS, phosphate buffered saline; S1P, sphingosine-1-phosphate; SPF, specific-
pathogen-free; TCR, T cell receptor
* E-mail: email@example.com (MAF); firstname.lastname@example.org (JAB)
. These authors contributed equally to this work.
Sphingolipids and their breakdown products modulate a variety
of eukaryotic signaling pathways involved in proliferation, apopto-
sis, differentiation, and migration (Figure 1). Although sphingolipids
are ubiquitous among eukaryotes, few bacteria produce them .
The genus Bacteroides and its relatives are an important exception;
40%–70% of the membrane phospholipids of these prominent
symbionts are sphingolipids [2,3]. While the structures of several
Bacteroides sphingolipids have been solved, the full repertoire of these
molecules has not yet been defined [1–19]. Here, by systematically
exploring the sphingolipid repertoire of Bacteroides fragilis, we show
that this gut commensal unexpectedly produces an isoform of a-
galactosylceramide, a sponge-derived sphingolipid that is the
prototypic ligand for the host immune receptor CD1d.
Results and Discussion
Bioinformatic Insights into Bacteroides fragilis
To gain insight into the potential role of Bacteroides sphingolipids
in mediating microbiota–host interactions, we set out to define the
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complete set of sphingolipids produced by Bacteroides fragilis NCTC
9343 , a genome-sequenced, genetically manipulable human
gut isolate. Reasoning that a chromatographic comparison of lipid
extracts from wild-type B. fragilis and a sphingolipid-deficient
mutant would reveal the complete set of B. fragilis sphingolipids, we
began by attempting to identify genes involved in B. fragilis
sphingolipid biosynthesis. We took a candidate gene approach,
hypothesizing that the Bacteroides sphingolipid pathway would
harbor homologs of the eukaryotic pathway . BLAST searches
of the B. fragilis genome using the Saccharomyces cerevisiae sphingo-
lipid biosynthetic enzymes as queries yielded two hits encoded by
adjacent genes: BF2461, a putative serine palmitoyltransferase,
and BF2462, a putative sphinganine kinase.
Bioinformatic analysis suggested that BF2461, like its yeast
homolog, is a pyridoxal-phosphate-dependent a-oxoamine syn-
thase that conjugates serine and a long-chain acyl-CoA to form 3-
dehydrosphinganine. In eukaryotes, this serves as the first
committed step in the sphingolipid biosynthetic pathway. We
therefore predicted that a D2461 mutant would be completely
deficient in the production of sphingolipids. The eukaryotic
homolog of BF2462, sphingosine kinase, phosphorylates sphingo-
sine to form sphingosine-1-phosphate (S1P). Given that this
reaction diverts the flux of the sphingosine base away from
ceramide and toward S1P, we hypothesized that a D2462 mutant
would produce a higher titer of mature sphingolipids than the
Using Genetics and Chemistry to Define the B. fragilis
We constructed a mutant harboring a deletion of BF2461
(D2461) (see S1.8 in Supporting Information S1). Although we
obtained co-integrates for the BF2462 mutant, double crossover
mutants were never obtained despite repeated attempts to screen
through thousands of colonies, suggesting that BF2462 may be
Bacteroides fragilis α-galactosylceramide
Agelas mauritianus α-galactosylceramide
Bacteroides fragilis ceramide phosphorylethanolamine
Bacteroides fragilis ceramide
Human plasma sphingomyelin
Figure 1. Chemical structures of the B. fragilis sphingolipids and related molecules. (A) B. fragilis produces the phosphosphingolipid
ceramide phosphoryl-ethanolamine (CPE, top) and the corresponding free ceramide (ceramideBf, middle), which are similar in structure to the most
abundant (4,5-dehydro) and third-most abundant (4,5-dihydro) forms of sphingomyelin in human plasma (bottom). (B) B. fragilis produces the
glycosphingolipid a-galactosylceramide (a-GalCerBf, top), which is similar in structure to the sponge-derived a-galactosylceramide agelasphin-9b
(middle) and a widely used derivative of agelasphin-9b, KRN7000 (bottom). Chemical groups that vary among the molecules in each column are
colored red and blue for B. fragilis and non–B. fragilis sphingolipids, respectively. CPE, ceramideBf, and a-GalCerBfwere each purified as inseparable
mixtures of varying lipid chain length. The proposed structures of the most abundant species are shown here.
While human gut bacteria are thought to produce
diffusible molecules that influence host biology, few of
these molecules have been identified. Species of Bacteroi-
des, a Gram-negative bacterial genus whose members
often comprise .50% of the gut community, are unusual
in that they produce sphingolipids, signaling molecules
that play a key role in modulating the host immune
response. Sphingolipid production is ubiquitous among
eukaryotes but present in only a few bacterial genera. We
set out to construct a Bacteroides strain that is incapable of
producing sphingolipids, knocking out a gene predicted to
encode the first enzymatic step in the Bacteroides
sphingolipid biosynthetic pathway. The resulting mutant
is indeed deficient in sphingolipid production, and we
purified and solved the structures of three sphingolipids
that are present in the wild-type strain but absent in the
mutant. To our surprise, one of these molecules is a close
chemical relative of a sponge sphingolipid that is the
prototypical ligand for a host receptor that controls the
activity of natural killer T cells. Like the sponge sphingo-
lipid, the Bacteroides sphingolipid can modulate natural
killer T cell activity, suggesting a novel mechanism by
which Bacteroides in the gut might influence the host
Bacteroides fragilis Produces a-Galactosylceramide
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essential for Bacteroides viability. An interesting alternative comes
from the observation that dihydrosphingosine, the putative
substrate of BF2462, is toxic to Bacteroides melaninogenicus at 4 mM
; the absence of BF2462 could therefore lead to the buildup of
a toxic intermediate.
Nevertheless, since the yeast homolog of BF2461 constitutes the
entry point to the sphingolipid pathway, we hypothesized that the
D2461 mutant would be sphingolipid-deficient, providing an ideal
starting point for enumerating the B. fragilis sphingolipids. To test
our hypothesis, we used comparative HPLC-ELSD to analyze
alkaline-stable lipid extracts from the wild-type (WT) and D2461
strains. Our analysis revealed three primary peaks that were
present in the WT but not the D2461 extract (Figure 2).
Preparative thin layer chromatography was used to purify
multimilligram quantities of these compounds, and HPLC-MS
analysis of the purified material revealed that each peak consists of
a mixture of co-migrating compounds that vary in mass by 14 Da.
Measured in negative mode, the most abundant mass ions for
peaks 1, 2, and 3 were 677.5 Da, 554.5 Da, and 716.6 Da,
Elucidating the Structures of the B. fragilis Sphingolipids
To solve the chemical structures of the sphingolipid species, we
first subjected the purified compounds to high-resolution MS. The
mass of peak 1 was consistent with ceramide phosphorylethano-
lamine (CPE) (C36H74N2O7P; [M-H]2m/z: calculated 677.5234,
observed 677.5221), a sphingomyelin isoform previously found to
be the principal B. fragilis sphingolipid, while the mass of peak 2
was consistent with the corresponding dihydroceramide base
m/z: calculated 554.5148, observed
554.5156) (Figure 1A; Figure S1 in Supporting Information S1).
A set of 1D and 2D NMR experiments on the purified compounds
from peaks 1 and 2 yielded resonances and couplings consistent
with these assignments (see S4.1 and S4.3 in Supporting
B. fragilis Produces a-Galactosylceramide
In contrast, peak 3 was not a known compound. High-
resolution MS analysis of the purified material from peak 3 was
consistent with an empirical formula of C40H79NO9([M-H]2m/z:
calculated 716.5682, observed 716.5698). 2D NMR analysis
indicated that this compound and CPE harbor an identical
dihydroceramide base (C34H68NO4), suggesting that the difference
(C6H11O5) corresponded to a distinct head group. Four lines of
evidence suggest that this head group is an a-configured galactose:
(i) The molecular formula is consistent with a glycosphingolipid
bearing a hexose as a head group. (ii) MS/MS analysis reveals a
fragment that is consistent with the elimination of a hexose head
group from a ceramide base ([M-H]2m/z: calculated 536.5048,
observed 536.5055). (iii) The
anomeric proton with a chemical shift of 4.64, consistent with
an a-linkage. (iv) Chemically synthesized a-galactosylceramide,
prepared by selective a-galactosylation of the B. fragilis dihydro-
ceramide base (see S1.10 in Supporting Information S1), has a1H
NMR spectrum indistinguishable from that of peak 3 (see S4.2 in
Supporting Information S1). We term this novel glycosphingolipid
B. fragilis a-galactosylceramide (a-GalCerBf) (Figure 1B). a-
GalCerBf, CPE, and the ceramide base were each purified as an
inseparable mixture of varying lipid chain length. This inseparable
mixture of alpha-galactosylceramides, hereafter ‘‘purified a-
GalCerBf,’’ was the material used for the immunological exper-
iments described below.
a-GalCerBfis a close structural relative of the sponge-derived a-
galactosylceramide agelasphin-9b (Figure 1B) ; aside from a-
1H NMR spectrum shows an
GalCerBf and the sponge-derived agelasphins, no naturally
occurring a-galactosylceramides have ever been discovered.
Substantial data have accumulated suggesting that a-GalCer is a
ligand for a subset of human and mouse T cells, termed invariant
natural killer T cells (iNKT), which express a conserved T cell
receptor (TCR) that recognizes glycolipids presented by the major
histocompatibility complex class I-like molecule, CD1d . A
synthetic derivative of agelasphin-9b termed KRN7000 (Figure 1B)
is the prototypical agonist of iNKT cells and has become a
critically important reagent for studying NKT cell biology both in
vitro and in vivo. Indeed, iNKT cells are often identified or isolated
by flow cytometry on the basis of their ability to bind a synthetic
tetramer of CD1d loaded with a derivative of KRN7000. A variety
of iNKT cell ligands have been described. One class consists of
low-affinity host-derived self-ligands such as isoglobotrihexosylcer-
amide and b-glucopyranosylceramide [23,24]. Another class
includes glycolipids from bacterial species including GSL-1 from
Sphingomonas, BbGL-II from Borrelia, and a family of diacylglycerol-
containing glycolipids from Streptococcus pneumoniae, all of which
have been postulated to be naturally occurring ligands for CD1d
[25–27]. It has also been proposed that liver infection by
Novosphingobium aromaticivorans, a close relative of Sphingomonas that
produces CD1d-binding sphingolipids, results in an NKT-cell-
dependent autoimmune response against the liver and bile ducts
Purified a-GalCerBfBinds to CD1d and Stimulates Mouse
and Human iNKT Cells
Based on the striking chemical similarity of a-GalCerBf to
KRN7000, we reasoned that a-GalCerBf might serve as an
endogenous ligand for CD1d and stimulate iNKT cell activity. To
test our hypothesis, we began by loading synthetic mouse CD1d
tetramers with purified a-GalCerBfand determining the ability of
the sphingolipid/CD1d-tetramer complex (hereafter ‘‘tetramer’’)
to stain two iNKT-cell-derived hybridomas [29,30]. As with
KRN7000, the a-GalCerBf-loaded tetramer (but not an empty
tetramer) bound both hybridomas but not a CD4+MHCII
restricted hybridoma reactive to GFP (GFP-36) (manuscript in
preparation, Yadav and Bluestone), indicating that the tetramer
staining was ligand- and TCR-specific (Figure 3A; Figure S2 in
Supporting Information S1). The iNKT cell hybridomas tested
produced IL-2 in response to both the marine-sponge-derived and
B. fragilis-derived sphingolipids in a dose-dependent manner and in
absence of antigen presenting cells (APCs). These results suggested
that a-GalCerBf is a stimulatory ligand that directly activates
iNKT cells in vitro (Figure 3B–C; Figure S3 in Supporting
We next examined the ability of purified a-GalCerBf to
stimulate freshly isolated mouse and human iNKT cells in vitro
and in vivo. Liver mononuclear cells, 30%–50% of which are NKT
cells, were incubated with splenocytes as APCs in the presence of
increasing doses of a-GalCerBfand examined for IFN-c produc-
tion. a-GalCerBfinduced IFN-c in a dose-dependent and CD1d-
dependent manner. The response was inhibited completely by
anti-CD1d antibodies (Figure 3D), consistent with our previous
result that NKT cell stimulation required ligand presentation by
CD1d (Figure 3B).
To explore whether the response of NKT cells to a-GalCerBfis
conserved in humans, we determined whether Va24+cells could
be expanded in vitro with purified a-GalCerBf as previously
described for KRN7000 . We cultured peripheral blood
mononuclear cells (PBMCs) from six independent donors with
0.1 mg/ml KRN7000, 1 mg/ml a-GalCerBf, or 1 mg/ml cerami-
deBffor 13 d and assessed the presence of CD3+Va24+cells by
Bacteroides fragilis Produces a-Galactosylceramide
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elution time (min)
total ion count (x106)
elution time (min)
Bacteroides fragilis Produces a-Galactosylceramide
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flow cytometry (Figure 3E–F). PBMCs cultured with KRN7000 or
a-GalCerBfshowed an expansion of a population of CD3+Va24+
cells, while PBMCs left untreated or treated with ceramideBfdid
not show an expansion of this population. Importantly, this result
shows that the activity of a-GalCerBfis specific and not due to a
contaminant of the lipid purification process since ceramideBf,
which was purified in a similar manner, did not exhibit this effect.
These results demonstrate that a-GalCerBfhas similar activities in
murine and human NKT cells and binds human CD1d.
To test whether a-GalCerBfcan activate iNKT cells in vivo, mice
were immunized with BMDCs pulsed with LPS alone or LPS +
purified a-GalCerBf. Consistent with activation, iNKT cells
isolated from the liver showed upregulation of the cell surface
markers CD25 and CD69 (Figure 3G), 15% of these liver-resident
iNKT cells expressed IFN-c after treatment (Figure 3H), and
elevated IFN-c levels were observed in the serum of these mice
(Figure 3I). Anti-CD1d blocking antibodies inhibited liver iNKT
cell activation and IFN-c production, demonstrating the specificity
of iNKT cell activation (Figure 3G–I). We therefore conclude that
a-GalCerBf is capable of stimulating iNKT cell activation and
cytokine production in vivo.
A Physiological Context for the Activity of KRN7000
The marine sponge-derived agelasphins and the nonphysiolo-
gical CD1d ligand KRN7000 have been the basis for numerous
studies over the last two decades implicating iNKT cells in
immunity (‘‘a-galactosylceramide’’ has 3,290 citations in Google
Scholar, 5/29/12). Unlike the pathogens from which CD1d
ligands have previously been isolated, Bacteroides is extraordinarily
prevalent in the human population, comprising .50% of the
trillions of cells in the gut community of a typical human . By
showing that B. fragilis produces the only known a-galactosylcer-
amide other than the sponge-derived agelasphins, and demon-
strating that a-GalCerBfbinds to CD1d and activates iNKT cells in
vitro and in vivo, our results suggest a physiological basis for the
activity of KRN7000. It is tempting to speculate that CD1d and
iNKT cells function in the context of a microbiota–host
interaction, especially in light of a recent report showing that
neonatal colonization of germ-free mice by a conventional
microbiota downregulates the level of iNKT cells in the colonic
lamina propria and lung . Indeed, it has been hypothesized
that the agelasphins are not produced by Agelas mauritianus, but
instead by a bacterial symbiont that inhabits the sponge .
In an attempt to determine the in vivo effect a-GalCerBfon NKT
cells, we colonized germ-free (GF) mice with WT or sphingolipid-
deficient B. fragilis by gavage and measured the percent and
activation status of NKT cells in the liver and spleen. Colonization
was confirmed by fecal cultures and PCR. We varied the length of
colonization (1, 3, 4, and 14 d), the mice’s age at the time of
colonization (4 and 8 wk old), sex, and strain (Swiss Webster and
C57BL/6). Several of these experiments indicated an expansion of
NKT cells mice colonized by WT but not mutant B. fragilis.
However, the effect was inconsistent and the levels of NKT cells in
our control mice—germ-free (GF) and specific-pathogen-free
(SPF)—fluctuated widely. As a percentage of total liver lympho-
cytes in the GF mice, NKT cells (CD3+tetramer+) varied between
8% and 48%, making it difficult to draw any conclusions about
differences in NKT cell number or activation markers between our
experimental data points.
Blumberg and coworkers recently showed that GF mice have
increased levels of NKT cells in the colon compared to SPF mice
and that colonization of neonatal, but not adult, GF mice with
microbiota from SPF mice can reverse this effect . Interest-
ingly, neither the increase nor the reversal after colonization is
seen in the liver or the spleen and there were no changes in the
activation status of NKT cells. Taken together, our results suggest
that the microbiota may affect NKT cells in the colon but not the
liver or spleen, and that interventions to change the numbers of
NKT cells must occur very early in life and may take weeks to be
evident. Although Blumberg and coworkers showed the effects of
the microbiota on NKT cell numbers and morbidity in models of
IBD and allergic asthma, they did not identify the strain or the
molecular pathway responsible for these effects; our results raise
the possibility that a-GalCerBf, produced by B. fragilis, may be at
least partially responsible for the results seen in their models.
There are subtle but important differences between KRN7000
and a-GalCerBf, indicating that the natural ligands for CD1d may
be less potent than KRN7000. The principal structural differences
between a-GalCerBfand KRN7000 are (i) a shorter N-acyl chain
bearing a hydroxyl group on the b- rather than the a-carbon, (ii)
the absence of a hydroxyl group at C4 of the sphinganine base,
and (iii) iso-branched lipid termini (Figure 1B). Synthetic
derivatives of KRN7000 that either have shorter N-acyl chains
or lack a C4 hydroxyl group have been shown to have less potent
activity and/or an altered cytokine response, an effect that might
be due to a change in the conformation of the CD1d–lipid
complex . Notably, one of the iso-branched lipid termini of a-
GalCerBfis shared with agelasphin 9b. Since iso-branched lipids
are commonly associated with specific bacterial genera (for
example, comprising 55%–96% of the total fatty acid pool in
Bacteroides) , their presence in agelasphin 9b is consistent with a
bacterial origin for these sponge-derived sphingolipids.
A Proposed Pathway for Bacteroides Sphingolipid
The absence of CPE, dihydroceramide, and a-GalCerBffrom
the D2461 mutant confirms that BF2461 is involved in B. fragilis
sphingolipid biosynthesis, marking the first known member of the
Bacteroides sphingolipid pathway (Figure 4). BF2461 is widely
conserved among human-associated genera of Bacteroidales
including Bacteroides, Parabacteroides, Porphyromonas, and Prevotella
(known sphingolipid producers) but absent from Alistipes (a
nonproducer), supporting its role in the bacterial sphingolipid
pathway. Our inability to construct a deletion mutant of BF2462
prevents us from exploring its potential role in the pathway,
though it is tempting to speculate that it generates dihydro-
sphingosine-1-phosphate from dihydrosphingosine. Although the
later steps of the pathway remain unclear, the intermediacy of
dihydroceramide is supported by the fact that CPE and a-
GalCerBf share a common C34 scaffold and by our direct
Figure 2. B. fragilis D2461 is deficient in the production of sphingolipids. HPLC-MS traces of crude lipid extracts of (A) wild-type B. fragilis and
(B) the sphingolipid-deficient mutant DBF2461 are shown. The traces shown are the total ion count (black) and the extracted ion traces of sphingolipid
masses for ceramide (m/z [M-H]: 540.5, 554.5, 568.5, 582.6; green), CPE (m/z [M-H]: 663.5, 677.5, 691.5, 705.5; brown), a-GalCerBf(m/z [M-H]: 702.6, 716.6,
730.6, 744.6; blue), and phosphatidylethanolamine (m/z [M-H]: 648.5, 662.5, 676.5, 690.5). Peaks corresponding to the three sphingolipids, but not the
phospholipid phosphatidylethanolamine, are absent in B. fragilis D2461. (C) High-resolution mass spectra of CPE, ceramideBf, and a-GalCerBfcollected in
the negative ion mode. The insets show a zoomed-in view of the dominant field of peaks for each compound. (D) A table showing the calculated and
observed masses for the dominant mass ions for each compound. See S1.1 in Supporting Information S1 for details.
Bacteroides fragilis Produces a-Galactosylceramide
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observation of dihydroceramide production by B. fragilis. On the
basis of these observations, we propose a model of Bacteroides
sphingolipid biosynthesis that closely mirrors the eukaryotic
pathway (Figure 4). Given that sphingolipids comprise ,30% of
total cellular lipids and Bacteroides lacks an endoplasmic reticulum
(the site of eukaryotic sphingolipid synthesis), the regulation of this
pathway in the context of lipid metabolism and the localization of
its biosynthetic enzymes will be important areas to explore.
Figure 3. a-GalCerBfbinds CD1d and activates NKT cells. (A) Hybridomas were stained with anti-CD3 antibodies and empty mCD1d tetramers
or CD1d tetramers loaded with a-GalCerBfor KRN7000. Flow cytometry plots are pregated on DAPI2events in lymphocyte gate stained with CD3
antibodies and the specified tetramer. Plots representative of three independent experiments are shown. (B) Hybridomas were cultured with BMDCs
pre-pulsed with LPS or LPS + a-GalCerBfin the presence of control Ig or anti-CD1d blocking antibodies. IL-2 secretion was measured in supernatants
16 h later. Data are representative of three independent experiments. (C) Plates were coated with CD1d monomers and loaded with the specified
amounts of a-GalCerBf. Hybridomas were then incubated for 16–18 h and IL-2 was measured in the supernatants by ELISA. Data are representative of
three independent experiments. (D) Liver mononuclear cells were cultured with splenocytes plus increasing amounts of a-GalCerBfin the presence or
absence of anti-CD1d blocking antibodies. IFN-c secretion was measured in supernatants on day 5. Data are representative of three independent
experiments. (E and F) Representative flow cytometry plots and pooled data of PBMCs cultured for 13–14 d with 0.1 mg/ml KRN7000, 1 mg/ml a-
GalCerBf, or 1 mg/ml ceramideBf. Dot plots show all events in the lymphocyte gate stained with 6B11 (specific for Va24) and CD3 antibodies. Gate
shows percentage of Va24+CD3+NKT cells pre- and postexpansion. Pooled data showing six individual donors tested in three independent
experiments. *p=0.0078, **p=0.0020 compared to control day 13 culture. (G–I) Bone-marrow-derived dendritic cells were pulsed in vitro with LPS
only or LPS + a-GalCerBffor 24 h. The 0.46106cells were transferred to WT mice, which were treated with control Ig or anti-CD1d blocking antibody
prior to cell transfer. Liver mononuclear cells were analyzed 16–18 h later. Data shown were pooled from three independent experiments. (G)
Expression of CD25 and CD69 on gated CD3+tetramer+cells. Representative flow cytometry plots and pooled data showing fold change of CD25 and
CD69 surface expression compared to NKT cells isolated from mice transferred with LPS-pulsed BMDCs. (H) Representative flow cytometry plots and
pooled data of intracellular IFN-c expression on gated CD3+tetramer+cells. (I) Serum IFN-c levels.
Bacteroides fragilis Produces a-Galactosylceramide
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Materials and Methods
Detailed methods are provided in Supporting Information S1.
Construction of Mutant Strain DBF2461
Primer sequences are listed in Table S1 in Supporting Information
S1. DNA fragments flanking BF2461 were PCR amplified from B.
intotheSstIsite ofpNJR6.The resulting plasmid wasintroducedinto
B. fragilis NCTC9343 by conjugation, and cointegrates were selected
using erythromycin. Cointegrates were passaged, plated on nonse-
lective medium, and replica plated to medium containing erythro-
mycin. Erythromycin-sensitive colonies were screened by PCR to
detect those acquiring the mutant genotype.
Purification of a-GalCerBf
B. fragilis NCTC9343 was cultured under standard conditions,
and harvested cells were extracted with CHCl3:MeOH (2:1). The
organic extract was subjected to alkaline hydrolysis, neutralized,
and extracted with CHCl3:MeOH (2:1). The crude extract was
purified by preparative TLC (CHCl3:MeOH:H2O, 65:25:4) to
give a-GalCerBf (Rf=0.6). For complete experimental details,
including yields and full characterization (NMR, high-resolution
mass spectrometry) of all compounds, see Supporting Information
S1. a-GalCerBfwas isolated in five independent batches, and the
in vitro and in vivo experiments were repeated with different
batches of purified compound.
a-GalCerBfUsed for Immunological Experiments
a-GalCerBf, CPE, and the ceramide base were each purified as
an inseparable mixture of varying lipid chain length. Mass spec
analysis of the methanolyzed long chain base (LCB) (S4.6 in
Supporting Information S1) suggests that this portion of the
structure carries the variation (see next paragraph). The insepa-
rable mixture of alpha-galactosylceramides (.95% pure), referred
to as ‘‘purified a-GalCerBf,’’ was the material used for the
ceramide phosphorylethanolamine (CPE)
long chain acyl-CoA
Figure 4. Proposed pathway for Bacteroides sphingolipid biosynthesis. BF2461, a putative serine palmitoyltransferase, would catalyze the
pyridoxal-phosphate-dependent conjugation of serine and a long-chain acyl-CoA to form 3-ketodihydrosphingosine, which would undergo a
ketoreductase-catalyzed conversion to dihydrosphingosine. At this branchpoint, dihydrosphingosine could either be phosphorylated by the putative
sphingosine kinase BF2462 to form S1P, or it could undergo N-acylation to yield the observed dihydroceramide intermediate (compound 2). This
common C34 scaffold would then be the substrate for two alternative head group modifications: glycosylation to form a-GalCerBf, or
phosphorylethanolamine group transfer to form CPE.
Bacteroides fragilis Produces a-Galactosylceramide
PLOS Biology | www.plosbiology.org7 July 2013 | Volume 11 | Issue 7 | e1001610
Analysis of Lipid Tail Length Variation
Methanolysis of ceramideBfproduced a mixture of three LCB
amines that could be separated and analyzed by HPLC-MS (S4.6
in Supporting Information S1). Analysis of each by HRMS
indicated that they are structural variants that differ in tail chain
length. These data suggest the major parent a-GalCerBfvariants
(m/z 716.57, m/z 730.58, and m/z 744.60) also differ in chain
length of the LCB.
For dose titration experiments, BMDCs and DN3A4-1.2 and
N38-2C12 NKT hybridomas (M. Kronenberg) and GFP36 CD4+
hybridoma were cultured at a 3:1 hybridoma:BMDC ratio and the
indicated doses of KRN7000 or a-GalCerBfin the presence of
1 mg/ml LPS. Supernatants were harvested after 24 h and IL-2
production was measured by ELISA. For APC-free experiments,
CD1d monomers were coated on a 96-well plate for 1 h, and wells
were blocked with PBS/10% FBS. The indicated amount of a-
GalCerBfwas added to each well and incubated at 37uC for 3 h.
After washing unbound a-GalCerBf, hybridomas were added.
Supernatants were harvested after 16–18 h and IL-2 production
was measured by ELISA. For in vitro CD1d blocking experiments,
a-GalCerBf pulsed BMDCs were cultured at a 3:1 hybrido-
ma:BMDC ratio in the presence of 10 mg/mL anti-CD1d
antibody (Clone 1B1, BD Pharmingen). Supernatants were
harvested after 16–18 h and IL-2 production was measured by
In Vitro Stimulation of Human NKT Cells
For blood draws from healthy donors, informed consent was
obtained in accordance with approved University of California,
San Francisco IRB policies and procedures (IRB 10-02596).
PBMCs were cultured for 13–14 d in RPMI containing 10%
autologous serum plus lipids as described in Figure 3. On day 1 of
culture, 100 U/ml hIL-2 was added. Cultures were harvested on
day 13 or 14 and the percentage of CD3+Va24+NKT cells was
determined by flow cytometry after staining with CD3 and 6B11
In Vivo Activation of NKT Cells
Mice were sacrificed 16–18 h after transfer of 0.46106mature
CD86hiMHCIIhiBMDCs. Livers were cut into small pieces and
passed through a stainless mesh. Cells were resuspended in 40%
Percoll solution (GE Healthcare), underlaid with 60% Percoll
solution, and centrifuged at 2,300 rpm for 20 min at room
temperature. All isolations were performed in the presence of
brefeldin A (Sigma). After cell surface staining, cells were fixed in
Cytofix/Cytoperm (BD Biosciences) according to the manufac-
turer’s instructions and stained for intracellular cytokines. Serum
IFN-c was measured by ELISA.
Supporting Information S1
Section S1, materials, equipment, and general methods.
Section S2, high-resolution mass spectrometry and LC-MS analysis.
Section S3, in vitro titration data.
Section S4, spectral data.
Figure S1, B. fragilis D2461 is deficient in the production of
sphingolipids. LC-MS trace with extracted ions shown [M-H]. See
Figure 2 legend for details.
Figure S2, a-GalCerBf binds CD1d in vitro. Hybridomas were
stained with anti-CD3 antibodies and empty mCD1d tetramers or
mCD1d tetramers loaded with a-GalCerBf or KRN7000. Flow
cytometry plots representative of three independent experiments
are shown. (A) Plots show forward (FSC) and side (SSC) scatter of
all events. (B) Plots pre-gated as shown in (A) and further gated on
DAPI-negative events show staining with tetramer and CD3
Figure S3, KRN7000 and a-GalCerBftitration in vitro. BMDCs
and NKT hybridomas were cultured at a 3:1 hybridoma:BMDC
ratio and the indicated doses of KRN7000 or a-GalCerBfin the
presence of 1 mg/ml LPS. Supernatants were harvested after 24 h
and IL-2 production was measured by ELISA.
Table S1, Primers used in this study.
We are indebted to the Abbas Lab for providing GM-CSF and IL-4, to the
NIH tetramer facility for providing unloaded CD1d monomers and PBS-
57-loaded tetramers, to Shilpi Jayaswal and Amy Putnam for assistance
with the NKT cell cultures, and to Hans Dooms for a critical reading of the
manuscript. We acknowledge the support of TuKiet Lam in the FT-ICR
Mass Spectrometry Resource of the Keck Biotechnology Resource
Laboratory and Angela Hansen and Jonathan Karty in the Indiana
University Mass Spectrometry Facility for help with mass spectrometry.
The author(s) have made the following declarations about their
contributions: Conceived and designed the experiments: LCB CP PCK
BBW JC MK JLS LEC JAB MAF. Performed the experiments: LCB CP
PCK BBW. Analyzed the data: LCB CP PCK BBW JC MK JLS LEC JAB
MAF. Wrote the paper: LCB CP JAB MAF.
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